P1369: HDAC INHIBITION INVOLVES CD26 EXPRESSION ON MULTIPLE MYELOMA CELLS VIA THE C-MYC/SP1-MEDIATED PROMOTER ACTIVATION AND OVERCOMES THERAPEUTIC RESISTANCE BY HUMANIZED ANTIBODY
Authors: Hiroko Nishida, Reiko Suzuki, Kiyora Nakajima, Chikao Morimoto, Michiie Sakamoto
Journal: HemaSphere (2023)

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: CD26 is expressed on several tumor cells including malignant lymphoma has been implicated in tumorigenesis, whereas, its roles in plasma cell malignancies remain elusive. Recently, we have identified that CD26 is uniformly and intensely expressed in osteoclasts, whereas its expression in plasma cells of multiple myeloma (MM) patients reveals heterogenous, leading to a marked difference of response to humanized anti-CD26 mAb in MM. Decreased expression levels of CD26 in myeloma cells are one of the mechanisms underlying resistance to CD26mAb therapy in MM. Aims: We elucidated the impacts and mechanisms of HDAC inhibition (HDACi) on the regulation of CD26 expression on myeloma cells, thereby eliciting anti-myeloma efficacy by CD26mAb in MM. Methods:Results: Immunostaining of bone marrow tissues of MM showed that CD26pos/CD138pos plasma cells were detected in several patients but not in others. First, we investigated the impact of HDACi on CD26 expression of myeloma cells. Although the cell surface expression of CD26 was relatively low or not detected on 5 myeloma cell lines: KMS11, 26, 27, 28, RPMI8226, cultured alone, an increase in CD26 expression levels was observed within 24 hrs of the treatment with broad inhibitors: panobinistat, vorinostat or isoform-selective inhibitors: romidepsin (HDAC1i), BG45, entinostat, RG2833 (HDAC3i) and nexturastat A, tubastatin A, ricolinostat (HDAC6i) by flow cytometry. CD26 levels increased further while exposure to each HDACi continued and the maximum increase was observed at 48 to 72 hrs. Subsequent removal of HDACi for 48 hrs resulted in a decline of CD26 expression levels on myeloma cells to baseline levels. Moreover, the CD26mRNA levels in myeloma cells were significantly increased by each HDACi. Next, we examined the impact of HDACi on the viability of CD26neg myeloma cells in the presence of CD26mAb. CD26mAb alone did not induce significant lysis of CD26neg myeloma cells, whereas, combining each HDACi plus CD26mAb synergistically facilitated lysis of CD26neg myeloma cells via direct effect as well as NK cell- mediated ADCC by CD26mAb. Of note, HDACi plus CD26mAb in combination readily augmented lysis of CD26neg cell populations, refractory to HDACi or CD26mAb alone. Transcriptome profiles in 3 myeloma cell lines in response to treatments with panobinostat or RG2833, compared with control IgG1 revealed that Myc is one of the most significantly down-regulated and overlapped genes. The 5’-flanking region of human CD26 gene, representing the promoter activity contains binding sites for the transcriptional factor, Sp1 which regulates the expression of c-Myc. Further, we explored the epigenetic impacts of c-Myc on the CD26 promoter of myeloma cells by HDACi. First, the expression level of c-Myc in KMS11, 27 and RPMI8226 was time-dependently decreased on treatment with panobinostat or RG2833 both at mRNA and protein levels. On the other hand, the acetylation of c-Myc on lysine 323 was significantly increased after 12 to 24 hr of exposure to each HDACi. ChIP-qPCR revealed that the binding of c-Myc to the CD26 promoter via the proximal G-C box was time-dependently reduced, whereas that of acetylated c-Myc on K323 to the promoter showed an increase in myeloma cells by each HDACi. Namely, in the absence of HDACi, c-Myc is attached to the CD26 promoter via binding to Sp1 and represses the promoter, leading to interruption of CD26 transcription in myeloma cells. In contrast, in the presence of HDACi, c-Myc is detached from the CD26 promoter via Sp1 with the increased acetylation of c-MycK323 and the promoter is activated, leading to initiation of CD26 transcription in myeloma cells. Conclusion: HDACi sensitizes myeloma cells with CD26 antigen loss to CD26mAb via c-Myc/Sp1-mediated CD26 induction and augments its cytotoxicity. Keywords: Molecular markers, Multiple myeloma, MYC, Monoclonal antibody